CA2103646A1 - Method and standard solution for the determination of thyroxine (t4) or triiodothyronine (t3) - Google Patents
Method and standard solution for the determination of thyroxine (t4) or triiodothyronine (t3)Info
- Publication number
- CA2103646A1 CA2103646A1 CA002103646A CA2103646A CA2103646A1 CA 2103646 A1 CA2103646 A1 CA 2103646A1 CA 002103646 A CA002103646 A CA 002103646A CA 2103646 A CA2103646 A CA 2103646A CA 2103646 A1 CA2103646 A1 CA 2103646A1
- Authority
- CA
- Canada
- Prior art keywords
- standard solution
- thyroxine
- triiodothyronine
- tbg
- albumin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- General Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Pathology (AREA)
- Endocrinology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
A b s t r a c t A standard solution containing bovine thyroxine-binding globulin (TBG) and thyroxine or triiodothyronine dissolved in a buffer solution is used for calibration in a method for the determination of thyroxine (T4) or triiodothyronine (T3) in serum.
Description
- 210364~
D e s c r i p t i o n The invention concerns a method for the determination of thyroxine (T4) or triiodothyronine (T3) in serum using a standard solution as well as this standard solution.
The thyroid hormones are present in blood mainly in a protein-bound form. The most important carrier protein for this is thyroxine-binding globulin, which is denoted TBG, to which ca. 80 % of the total thyroxine and triiodothyronine present in blood is bound.
Approximately 5 to 10 ~ of the total thyroxine is present bou~d to albumin and about 10 to 15 % of the total thyroxine is present bound to pre-albumin. The totality of these proteins is ~lso referred to as thyroxine-binding protein (TBP). Only very small proportions of thyroxine and triiodothyronineare present in blood in a free for~; usually ca. 0.03 % of T4 or 0.3 % of T3. Only the released hormones are physiologically active whereas the bound hormones T3 and T4 circulate in the blood as a metabolically inert transport form and serve as a buffer to regulate the level. It is therefore important to determine the total thyroxine content as well as the proportion of free thyroxine and free triiodothyronine in blood since this represents the functional thyroid status independent of changes in the concentration or saturation of the thyroxine binding protein.
Various methods are known for the determination of total thyroxine (T4), free thyroxine (FT4) and triiodothyronine (T3). A difficulty in all of these known methods is the provision of a standard which is suitable for the calibration.
D e s c r i p t i o n The invention concerns a method for the determination of thyroxine (T4) or triiodothyronine (T3) in serum using a standard solution as well as this standard solution.
The thyroid hormones are present in blood mainly in a protein-bound form. The most important carrier protein for this is thyroxine-binding globulin, which is denoted TBG, to which ca. 80 % of the total thyroxine and triiodothyronine present in blood is bound.
Approximately 5 to 10 ~ of the total thyroxine is present bou~d to albumin and about 10 to 15 % of the total thyroxine is present bound to pre-albumin. The totality of these proteins is ~lso referred to as thyroxine-binding protein (TBP). Only very small proportions of thyroxine and triiodothyronineare present in blood in a free for~; usually ca. 0.03 % of T4 or 0.3 % of T3. Only the released hormones are physiologically active whereas the bound hormones T3 and T4 circulate in the blood as a metabolically inert transport form and serve as a buffer to regulate the level. It is therefore important to determine the total thyroxine content as well as the proportion of free thyroxine and free triiodothyronine in blood since this represents the functional thyroid status independent of changes in the concentration or saturation of the thyroxine binding protein.
Various methods are known for the determination of total thyroxine (T4), free thyroxine (FT4) and triiodothyronine (T3). A difficulty in all of these known methods is the provision of a standard which is suitable for the calibration.
2 ~
For the calibration it is necessary to produce human serum standards with particular thyroxine or triiodothyronine concentrations and a defined FT4 or FT3 content. Human serum has previously been used as starting material for this. Firstly all the thyroxine and triiodothyronine in a free as well as in a bound form has to be removed from the human serum and then the human serum treated in this way has to again be supplemented with defined amounts of thyroxine or triiodothyronine. Various methods are described in the literature for this purpose such as treatment with active charcoal, with ion exchangers or with carrier-bound anti-T4 or anti-T3 antibodies. These methods are all very complicated. A further problem is that each human serum has a different composition thus leading to large variations in the individual lots and as a consequence the reproducibility leaves much to be desired.
A further disadvantage of these known standard solutions for the determination of T3 and T4 is that these solutions, which have the composition of human serum, cannot be stored for longer periods since on the one hand the protein which is present is gradually denatured and on the other hand there is a shift in the set equilibrium between bound and free T3 and T4.
It is known from EP-A O 337 467 that in order to solve the above-mentioned problems a standard solution can be used for a method to determine thyroxine or triiodothyronine which is still stable after long storage and can be produced in a simple manner using relatively cheap startin~ materials by using at least one standard solution for the calibration that contains ~03~'16 TBG and thyroxine or triiodothyronine dissolved in a buffer solution.
It has now been found that a further quite considerable improvement of the stability on storage can be achieved if TBG from bovine serum is used in the aforementioned methods.
The invention therefore concerns a method for the determination of thyroxine (T4) or triiodothyronine (T3) in serum in which a standard solution is used for the calibration which contains thyroxine-binding globulin (TBG) and thyroxine or triiodothyronine dissolved in a buffer solution which is characterized in that bovine TBG is used.
Whereas when using the known methods the standard solution in liquid reagent form can only just achieve a minimum storage capability of 8 weeks and therefore has to be stored in a lyophilized form, the standard solution used according to the present invention is stable on storage in a liquid form for more than 10 times as long. According to the results obtained thus far the storage stability in a liquid form will attain at least 18 months and after this time period there is still a full binding capacity for T4 and T3 whereas the corresponding standard solution with human TBG even when stored below 10C already yields erroneous values which are 20 % too high after 12 weeks. Moreover the bovine serum used to isolate bovine TBG is much more readily obtainable than human serum.
The superior stability of the standard solution according to the present invention can be seen in 210~
Table 1. From this it is apparent that with human TBG
(H-TBG) the analyte stability decreases rapidly so that erroneously high values are found which already after 3 months are 20 % higher (i.e. 120 ~ of the initial value) than with a fresh solution. In contrast using the standard solution according to the present invention no change whatsoever can be observed even after 12 months i.e. the analytical values are unchanged and correct at 100 %. The stated contents in percent relate to the measured recovery of analyte compared to a fresh product. Here it should be noted that analyte bound to TBG, i.e. T4 or T3, is not detected at all and an increase in the value for T4 or T3 means that the binding to TBG has deteriorated thus yielding false values compared to fresh natural TBG. The limit which is still ~ust acceptable is a change of + 5 %.
Various methods are known for determining thyroxine and triiodothyronine. Methods of determination are for example described in Nuc. Compact 16 (1985), 321-327, J.
Clin. Immunoassay 7 (1984), 192-205, and J. Clin. Chem.
~lin. Biochem. 22 (1984), 895-904. Determination methods based on immunoassays are used especially for determining thyroid hormones.
In a known method fsr the determination of thyroxine or triiodothyronine in serum based on an immunoassay technique labelled T4 or T3 and T4 or T3 from the sample usually compete for a binding partner such as for example an anti-T4 antibody. By adjusting the reaction conditions such as the concentration of the individual components, incubation period etc., it is possible to either determine the total thyroxine or total triiodothyronine or the free thyroxine or free triiodothyronine. For this the amount of labelled T3 or 2~0364~
T4 bound to the anti-T4 or anti-T3 antibody or non-bound labelled T3 or T4 is determined via known detection reactions. After measuring the label the content of T3 or T4 can then be calculated by comparing the value obtained to values obtained with known T3 or T4 concentrations. For this it is necessary to establish a calibration curve in which the measurement signal (e.g.
the absorbance value) is plotted against the T3 or T4 concentration. In the case of thyroxine it is expedient to establish a calibration curve from six thyroxine solutions with different concentrations since there is no linear relationship which would allow a two-point standard measurement.
The method according to the present invention is suitable for the determination of free thyroxine (FT4 ), free triiodothyronine (FT3) as well as of total thyroxine (T4) and thyroxine (T3) in serum.
The thyroxine-binding bovine globulin can be isolated from bovine sera in a known manner. The standard solution used according to the present invention preferably contains 5 to 30 ~g/ml bovine TBG. It is particularly preferred to use bovine TBG in a physiological amount which is in the range from 9 to 20 ~g/ml TBG which for example can be gathered from the literature references Lab. Med. 6 (1982), 27-2~, and J.
Clin. Chem. Clin. Biochem. 23 (1985) 117-127.
If the standard solution is to be used to determine thyroxine it additionally contains thyroxine. The thyroxine is preferably present in an amount of 5 to 500 ng/ml. It is particularly preferable to add a physiological amount of thyroxine to the standard ~1~3~
solution i.e. an amount in the range from 5 to 300 ng/ml.
If the standard solution is to be used to determine triiodothyronine it additionally contains triiodothyronine. The triiodothyronine is preferably used in an amount of 0.5 to 12 ng/ml. It is particularly preferable to add triiodothyronine in a physiological amount i.e. in the range of 0.5 to 8 ng/ml.
The standard solution used for the method according to the present invention preferably also contains albumin which improves the stability of the solution in addition to bovine TBG and thyroxine or triiodothyronine in buffer solution. In this connection it is not necessary to add human serum albumin, which is of course suitable, to the standard solution preferred according to the present invention. Equally suitable are the universally and easily obtainable and much more preferable albumins, for example from bovine or horse serum. Human serum albumin, bovine serum albumin or horse serum albumin and in particular bovine serum albumin is preferably used as albumin. The albumin is preferably used in an amount of 40 to 80 mg/ml solution. It is particularly preferable to use albumin in a physiological amount which is in a range from 50 to 70 mg/ml.
In order to produce the standard solution, bovine TBG
and, if desired, albumin are dissolved in a buffer solution. All buffers which have a pH range of 6.0 to 8.0 are suitable as the buffer system, preferably those with a pH value of 6.5 to 7.5. GOOD buffers are preferably used, for example HEPES or TRIS buffer. The buffer concentration is not critical, the buffer is preferably used at a concentration of 30 to 150 mmolJl.
6~
The standard solution particularly preferably contains 50 to 100 mmol/l buffer.
Subsequently the thyroxine or triiodothyronine in appropriate concentrations is added to this solution. In this process an equilibrium is established between protein bound and free thyroid hormones which i5 similar to the equilibrium which is present in vivo in blood.
This solution is also stable over long periods of storage because of its composition. Since it is produced from standardized individual substances it always has a uniform composition and therefore yields reproducible values~
In order to establish a calibration curve, several standard solutions - as a rule four to six - are used with different contents of thyroid hormones so that a curve can be drawn from the values obtained. In a preferred embodiment of the method according to the present invention a solution is used as the first standard solution which only contains bovine TBG and, if desired, albumin in a buffer solution but contains no thyroid hormone. Solutions with defined hormone contents are then used for the further calibration.
The invention also concerns a standard solution for the determination of thyroxine or triiodothyronine which contains bovine TBG and thyroxine or triiodothyronine dissolved in a buffer system.
The standard solution preferably contains 5 to 30 ~g/ml bovine TBG. It is particularly preferred that a physiological amount Df bovine TBG, i.e. 9 to 20 ~g/ml, is present in the standard solution.
36~
In a preferred embodiment the standard solution additionally contains albumin for stabilization. In this case the albumin is preferably present in an amount of 40 to 80 mg/ml. It is particularly preferable to use physiological amounts of albumin i.e. in the range of 50 to 70 mg/ml albumin.
~he standard solution is produced by dissolving bovine TBG and, if desired, albumin in a buffer system and adding the amount of thyroxine or triiodothyronine desired in each case. In this connection the amount of thyroxine is preferably in the range of 5 to 500 ng/ml and particularly preferably in the physiological range of 5 to 300 ng/ml. If the standard solution is used to determine triiodothyronine it then contains triiodothyronine prefe~ably in an amount of 0.5 to 12 ng/ml and particularly preferably in a physiological amount in the range of 0.5 to 8 ng/ml.
The buffer system preferably has a pH in the range 6.0 to 8.0 and is preferably a GOOD buffer.
According to the invention a standard solution with a superior stability is provided which can be produced in a simple manner from easily obtainable starting materials.
~3~4~
_ 9 _ Example Isolation of bovine TBG
Bovine plasma is brought to a concentration of 2 mol/l ammonium sulfate, the supernatant is decanted off diluted to 1 mol/l ammonium sulfate and applied to a chromatography column (Sepharose to which T4 is bound).
It is washed in succession with:
1. 50 mmol/l Tris pH 7.5, 100 mmol/l sodium chloride 2. 50 mmol/l Tris pH 7.5, 750 mmol/l sodium chloride 3. 50 mmol/l Tris pH 7.5, 100 mmol/l sodium chloride 4. 50 mmol/l Tris pH 7.5, 100 mmol/l sodium chloride 5 mmol/l 8-anilino-naphthaline sulfonic acid.
The eluate is concentrated in an osmotic cell and 8-anilino-naphthaline sulfonic acid is removed by dialysing for two days against 5 mmol/1 sodium phosphate pH 7.5, 10 mmol/l sodium chloride.
Example 2 Stability of T3/T4 calibrators Composition of the calibrators:
For the calibration it is necessary to produce human serum standards with particular thyroxine or triiodothyronine concentrations and a defined FT4 or FT3 content. Human serum has previously been used as starting material for this. Firstly all the thyroxine and triiodothyronine in a free as well as in a bound form has to be removed from the human serum and then the human serum treated in this way has to again be supplemented with defined amounts of thyroxine or triiodothyronine. Various methods are described in the literature for this purpose such as treatment with active charcoal, with ion exchangers or with carrier-bound anti-T4 or anti-T3 antibodies. These methods are all very complicated. A further problem is that each human serum has a different composition thus leading to large variations in the individual lots and as a consequence the reproducibility leaves much to be desired.
A further disadvantage of these known standard solutions for the determination of T3 and T4 is that these solutions, which have the composition of human serum, cannot be stored for longer periods since on the one hand the protein which is present is gradually denatured and on the other hand there is a shift in the set equilibrium between bound and free T3 and T4.
It is known from EP-A O 337 467 that in order to solve the above-mentioned problems a standard solution can be used for a method to determine thyroxine or triiodothyronine which is still stable after long storage and can be produced in a simple manner using relatively cheap startin~ materials by using at least one standard solution for the calibration that contains ~03~'16 TBG and thyroxine or triiodothyronine dissolved in a buffer solution.
It has now been found that a further quite considerable improvement of the stability on storage can be achieved if TBG from bovine serum is used in the aforementioned methods.
The invention therefore concerns a method for the determination of thyroxine (T4) or triiodothyronine (T3) in serum in which a standard solution is used for the calibration which contains thyroxine-binding globulin (TBG) and thyroxine or triiodothyronine dissolved in a buffer solution which is characterized in that bovine TBG is used.
Whereas when using the known methods the standard solution in liquid reagent form can only just achieve a minimum storage capability of 8 weeks and therefore has to be stored in a lyophilized form, the standard solution used according to the present invention is stable on storage in a liquid form for more than 10 times as long. According to the results obtained thus far the storage stability in a liquid form will attain at least 18 months and after this time period there is still a full binding capacity for T4 and T3 whereas the corresponding standard solution with human TBG even when stored below 10C already yields erroneous values which are 20 % too high after 12 weeks. Moreover the bovine serum used to isolate bovine TBG is much more readily obtainable than human serum.
The superior stability of the standard solution according to the present invention can be seen in 210~
Table 1. From this it is apparent that with human TBG
(H-TBG) the analyte stability decreases rapidly so that erroneously high values are found which already after 3 months are 20 % higher (i.e. 120 ~ of the initial value) than with a fresh solution. In contrast using the standard solution according to the present invention no change whatsoever can be observed even after 12 months i.e. the analytical values are unchanged and correct at 100 %. The stated contents in percent relate to the measured recovery of analyte compared to a fresh product. Here it should be noted that analyte bound to TBG, i.e. T4 or T3, is not detected at all and an increase in the value for T4 or T3 means that the binding to TBG has deteriorated thus yielding false values compared to fresh natural TBG. The limit which is still ~ust acceptable is a change of + 5 %.
Various methods are known for determining thyroxine and triiodothyronine. Methods of determination are for example described in Nuc. Compact 16 (1985), 321-327, J.
Clin. Immunoassay 7 (1984), 192-205, and J. Clin. Chem.
~lin. Biochem. 22 (1984), 895-904. Determination methods based on immunoassays are used especially for determining thyroid hormones.
In a known method fsr the determination of thyroxine or triiodothyronine in serum based on an immunoassay technique labelled T4 or T3 and T4 or T3 from the sample usually compete for a binding partner such as for example an anti-T4 antibody. By adjusting the reaction conditions such as the concentration of the individual components, incubation period etc., it is possible to either determine the total thyroxine or total triiodothyronine or the free thyroxine or free triiodothyronine. For this the amount of labelled T3 or 2~0364~
T4 bound to the anti-T4 or anti-T3 antibody or non-bound labelled T3 or T4 is determined via known detection reactions. After measuring the label the content of T3 or T4 can then be calculated by comparing the value obtained to values obtained with known T3 or T4 concentrations. For this it is necessary to establish a calibration curve in which the measurement signal (e.g.
the absorbance value) is plotted against the T3 or T4 concentration. In the case of thyroxine it is expedient to establish a calibration curve from six thyroxine solutions with different concentrations since there is no linear relationship which would allow a two-point standard measurement.
The method according to the present invention is suitable for the determination of free thyroxine (FT4 ), free triiodothyronine (FT3) as well as of total thyroxine (T4) and thyroxine (T3) in serum.
The thyroxine-binding bovine globulin can be isolated from bovine sera in a known manner. The standard solution used according to the present invention preferably contains 5 to 30 ~g/ml bovine TBG. It is particularly preferred to use bovine TBG in a physiological amount which is in the range from 9 to 20 ~g/ml TBG which for example can be gathered from the literature references Lab. Med. 6 (1982), 27-2~, and J.
Clin. Chem. Clin. Biochem. 23 (1985) 117-127.
If the standard solution is to be used to determine thyroxine it additionally contains thyroxine. The thyroxine is preferably present in an amount of 5 to 500 ng/ml. It is particularly preferable to add a physiological amount of thyroxine to the standard ~1~3~
solution i.e. an amount in the range from 5 to 300 ng/ml.
If the standard solution is to be used to determine triiodothyronine it additionally contains triiodothyronine. The triiodothyronine is preferably used in an amount of 0.5 to 12 ng/ml. It is particularly preferable to add triiodothyronine in a physiological amount i.e. in the range of 0.5 to 8 ng/ml.
The standard solution used for the method according to the present invention preferably also contains albumin which improves the stability of the solution in addition to bovine TBG and thyroxine or triiodothyronine in buffer solution. In this connection it is not necessary to add human serum albumin, which is of course suitable, to the standard solution preferred according to the present invention. Equally suitable are the universally and easily obtainable and much more preferable albumins, for example from bovine or horse serum. Human serum albumin, bovine serum albumin or horse serum albumin and in particular bovine serum albumin is preferably used as albumin. The albumin is preferably used in an amount of 40 to 80 mg/ml solution. It is particularly preferable to use albumin in a physiological amount which is in a range from 50 to 70 mg/ml.
In order to produce the standard solution, bovine TBG
and, if desired, albumin are dissolved in a buffer solution. All buffers which have a pH range of 6.0 to 8.0 are suitable as the buffer system, preferably those with a pH value of 6.5 to 7.5. GOOD buffers are preferably used, for example HEPES or TRIS buffer. The buffer concentration is not critical, the buffer is preferably used at a concentration of 30 to 150 mmolJl.
6~
The standard solution particularly preferably contains 50 to 100 mmol/l buffer.
Subsequently the thyroxine or triiodothyronine in appropriate concentrations is added to this solution. In this process an equilibrium is established between protein bound and free thyroid hormones which i5 similar to the equilibrium which is present in vivo in blood.
This solution is also stable over long periods of storage because of its composition. Since it is produced from standardized individual substances it always has a uniform composition and therefore yields reproducible values~
In order to establish a calibration curve, several standard solutions - as a rule four to six - are used with different contents of thyroid hormones so that a curve can be drawn from the values obtained. In a preferred embodiment of the method according to the present invention a solution is used as the first standard solution which only contains bovine TBG and, if desired, albumin in a buffer solution but contains no thyroid hormone. Solutions with defined hormone contents are then used for the further calibration.
The invention also concerns a standard solution for the determination of thyroxine or triiodothyronine which contains bovine TBG and thyroxine or triiodothyronine dissolved in a buffer system.
The standard solution preferably contains 5 to 30 ~g/ml bovine TBG. It is particularly preferred that a physiological amount Df bovine TBG, i.e. 9 to 20 ~g/ml, is present in the standard solution.
36~
In a preferred embodiment the standard solution additionally contains albumin for stabilization. In this case the albumin is preferably present in an amount of 40 to 80 mg/ml. It is particularly preferable to use physiological amounts of albumin i.e. in the range of 50 to 70 mg/ml albumin.
~he standard solution is produced by dissolving bovine TBG and, if desired, albumin in a buffer system and adding the amount of thyroxine or triiodothyronine desired in each case. In this connection the amount of thyroxine is preferably in the range of 5 to 500 ng/ml and particularly preferably in the physiological range of 5 to 300 ng/ml. If the standard solution is used to determine triiodothyronine it then contains triiodothyronine prefe~ably in an amount of 0.5 to 12 ng/ml and particularly preferably in a physiological amount in the range of 0.5 to 8 ng/ml.
The buffer system preferably has a pH in the range 6.0 to 8.0 and is preferably a GOOD buffer.
According to the invention a standard solution with a superior stability is provided which can be produced in a simple manner from easily obtainable starting materials.
~3~4~
_ 9 _ Example Isolation of bovine TBG
Bovine plasma is brought to a concentration of 2 mol/l ammonium sulfate, the supernatant is decanted off diluted to 1 mol/l ammonium sulfate and applied to a chromatography column (Sepharose to which T4 is bound).
It is washed in succession with:
1. 50 mmol/l Tris pH 7.5, 100 mmol/l sodium chloride 2. 50 mmol/l Tris pH 7.5, 750 mmol/l sodium chloride 3. 50 mmol/l Tris pH 7.5, 100 mmol/l sodium chloride 4. 50 mmol/l Tris pH 7.5, 100 mmol/l sodium chloride 5 mmol/l 8-anilino-naphthaline sulfonic acid.
The eluate is concentrated in an osmotic cell and 8-anilino-naphthaline sulfonic acid is removed by dialysing for two days against 5 mmol/1 sodium phosphate pH 7.5, 10 mmol/l sodium chloride.
Example 2 Stability of T3/T4 calibrators Composition of the calibrators:
6 % bovine serum albumin, 50 mmol/l Hepes buffer, pH 7.4 15 ~g/ml bovine TBG
FT3 content: 0, 2, 7, 15, 30 pgJml or FT4 content: 1, 10, 20, 40, 80 pg/ml ~lQ3~
The following Table shows the stability of T3/T4 calibrators.
Table 1:
Storage period Recovery1) with at 4 - 8C an analyte content (months) of 2 - 30 pg/ml .
T3 without TBG 3 118 T3 with human TBG 3 115 T3 with bovine TBG 3 100 1) The recovery shows the measurement value found for T3 in relation to the measurement value for T3 at the beginning of storage (100 ~).
FT3 content: 0, 2, 7, 15, 30 pgJml or FT4 content: 1, 10, 20, 40, 80 pg/ml ~lQ3~
The following Table shows the stability of T3/T4 calibrators.
Table 1:
Storage period Recovery1) with at 4 - 8C an analyte content (months) of 2 - 30 pg/ml .
T3 without TBG 3 118 T3 with human TBG 3 115 T3 with bovine TBG 3 100 1) The recovery shows the measurement value found for T3 in relation to the measurement value for T3 at the beginning of storage (100 ~).
Claims (37)
1. A method for the determination of thyroxine (T4) or triiodothyronine (T3) in serum whereby a standard solution is used for the calibration which contains thyroxine-binding globulin (TBG) and thyroxine or triiodothyronine dissolved in a buffer solution, wherein said TBG is bovine TBG.
2. A method as claimed in claim 1, wherein a solution is used as a further standard solution which contains bovine TBG dissolved in a buffer solution but contains no thyroxine or triiodothyronine.
3. A method as claimed in claim 1 or 2, wherein said standard solution further contains albumin.
4. A method as claimed in claim 3, wherein said albumin is bovine serum albumin.
5. A method as claimed in claim 3, wherein said standard solution contains 40 to 80 mg/ml of said albumin.
6. A method as claimed in claim 4, wherein said standard solution contains 40 to 80 mg/ml of said albumin.
7. A method as claimed in claim 3, wherein said standard solution contains 50 to 70 mg/ml of said albumin.
8. A method as claimed in claim 4, wherein said standard solution contains 50 to 70 mg/ml of said albumin.
9. A method as claimed in claim 1, 2, 4, 5, 6, 7 or 8, wherein said standard solution contains 5 to 30 µg/ml of said bovine TBG.
10. A method as claimed in claim 1, 2, 4, 5, 6, 7 or 8, wherein said standard solution contains 9 to 20 µg/ml of said bovine TBG.
11. A method as claimed in claim 1, 4, 5, 6, 7 or 8, wherein said standard solution contains 5 to 500 ng/ml of thyroxine.
12. A method as claimed in claim 9, wherein said standard solution contains 5 to 500 ng/ml of thyroxine.
13. A method as claimed in claim 10, wherein said standard solution contains 5 to 500 ng/ml of thyroxine.
14. A method as claimed in claim 1, 4, 5, 6, 7 or 8, wherein said standard solution contains 5 to 300 ng/ml of thyroxine.
15. A method as claimed in claim 9, wherein said standard solution contains 5 to 300 ng/ml of thyroxine.
16. A method as claimed in claim 10, wherein said standard solution contains 5 to 300 ng/ml of thyroxine.
17. A method as claimed in claim 1, 4, 5, 6, 7 or 8, wherein said standard solution contains 0.5 to 12 ng/ml of triiodothyronine.
18. A method as claimed in claim 9, wherein said standard solution contains 0.5 to 12 ng/ml of triiodothyronine.
19. A method as claimed in claim 10, wherein said standard solution contains 0.5 to 12 ng/ml of triiodothyronine.
20. A method as claimed in claim 1, 4, 5, 6, 7 or 8, wherein said standard solution contains 0.5 to 8 ng/ml of triiodothyronine.
21. A method as claimed in claim 9, wherein said standard solution contains 0.5 to 8 ng/ml of triiodothyronine.
22. A method as claimed in claim 10, wherein said standard solution contains 0.5 to 8 ng/ml of triiodothyronine.
23. A method as claimed in claim 1, 2, 4, 5, 6, 7, 8, 12, 13, 15, 16, 18, 19, 21 or 22, wherein said buffer solution has a pH range from 6 to 8.
24. A method as claimed in claim 1, 2, 4, 5, 6, 7, 8, 12, 13, 15, 16, 18, 19, 21 or 22, wherein said buffer solution contains a GOOD buffer.
25. A method as claimed in claim 1, 2, 4, 5, 6, 7, 8, 12, 13, 15, 16, 18, 19, 21 or 22, wherein said buffer solution contains 30 to 150 mmol/l buffer.
26. In a method for the determination of thyroxine (T4) or triiodothyronine (T3) in serum in which a calibration is carried out with a standard solution containing thyroxine-binding globulin (TBG) and thyroxine or triiodothyronine in a buffer solution, the improvement wherein said TBG is bovine TBG.
27. A standard solution for the determination of thyroxine or triiodothyronine containing bovine TBG
and thyroxine or triiodothyronine dissolved in a buffer solution.
and thyroxine or triiodothyronine dissolved in a buffer solution.
28. A standard solution as claimed in claim 27, wherein it contains 5 to 30 µg/ml bovine TBG and 30 to 150 mmol/l buffer.
29. A standard solution as claimed in claim 27 or 28, further containing albumin.
30. A standard solution as claimed in claim 29, wherein said albumin is present in an amount of 40 to 80 mg/ml.
31. A standard solution as claimed in claim 27, 28 or 30, which contains 5 to 500 ng/ml thyroxine.
32. A standard solution as claimed in claim 29, which contains 5 to 500 ng/ml thyroxine.
33. A standard solution as claimed in claim 27, 28 or 30, which contains 5 to 300 mg/ml thyroxine.
34. A standard solution as claimed in claim 27, 28 or 30, which contains 0.5 to 12 ng/ml triiodothyronine.
35. A standard solution as claimed in claim 29, which contains 0.5 to 12 ng/ml triiodothyronine.
36. A standard solution as claimed in claim 27, 28 or 30, which contains 0.5 to 8 ng/ml triiodothyronine.
37. A standard solution as claimed in claim 29, which contains 0.5 to 8 ng/ml triiodothyronine.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4226949A DE4226949A1 (en) | 1992-08-14 | 1992-08-14 | Method and standard solution for the determination of thyroxine (T¶4¶) or triiodothyronine (T¶3¶) |
| DEP4226949.0 | 1992-08-14 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2103646A1 true CA2103646A1 (en) | 1994-02-15 |
Family
ID=6465564
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002103646A Abandoned CA2103646A1 (en) | 1992-08-14 | 1993-08-09 | Method and standard solution for the determination of thyroxine (t4) or triiodothyronine (t3) |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0583717B1 (en) |
| JP (1) | JP2542787B2 (en) |
| KR (1) | KR940004326A (en) |
| AT (1) | ATE164229T1 (en) |
| AU (1) | AU656479B2 (en) |
| CA (1) | CA2103646A1 (en) |
| DE (2) | DE4226949A1 (en) |
| ES (1) | ES2115703T3 (en) |
| IL (1) | IL106634A0 (en) |
| ZA (1) | ZA935914B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE60130145T2 (en) * | 2000-12-21 | 2008-05-15 | Resuscitation Technologies, LLC, Santa Monica | STABLE T3 COMPOSITIONS AND ITS APPLICATION |
| EP3315966A4 (en) * | 2015-06-29 | 2018-11-14 | Fujirebio Inc. | Standard thyroid gland hormone solution |
| CN108776218A (en) * | 2018-05-31 | 2018-11-09 | 湖南远璟生物技术有限公司 | A kind of total triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4052504A (en) * | 1976-06-30 | 1977-10-04 | Corning Glass Works | Assay for thyroxine binding globulin |
| DE3812609A1 (en) * | 1988-04-15 | 1989-11-09 | Boehringer Mannheim Gmbh | METHOD FOR DETERMINING THYROXIN AND STANDARD SOLUTION SUITABLE FOR THIS |
| DE3812610A1 (en) * | 1988-04-15 | 1989-10-26 | Boehringer Mannheim Gmbh | METHOD FOR DETERMINING THE THYROXIN BINDING CAPACITY AND SUITABLE STANDARD SOLUTION |
-
1992
- 1992-08-14 DE DE4226949A patent/DE4226949A1/en not_active Withdrawn
-
1993
- 1993-08-09 IL IL106634A patent/IL106634A0/en unknown
- 1993-08-09 AU AU44534/93A patent/AU656479B2/en not_active Ceased
- 1993-08-09 CA CA002103646A patent/CA2103646A1/en not_active Abandoned
- 1993-08-10 EP EP93112770A patent/EP0583717B1/en not_active Expired - Lifetime
- 1993-08-10 DE DE59308274T patent/DE59308274D1/en not_active Expired - Fee Related
- 1993-08-10 AT AT93112770T patent/ATE164229T1/en not_active IP Right Cessation
- 1993-08-10 ES ES93112770T patent/ES2115703T3/en not_active Expired - Lifetime
- 1993-08-11 KR KR1019930015522A patent/KR940004326A/en not_active Application Discontinuation
- 1993-08-13 ZA ZA935914A patent/ZA935914B/en unknown
- 1993-08-13 JP JP5201395A patent/JP2542787B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| IL106634A0 (en) | 1993-12-08 |
| ES2115703T3 (en) | 1998-07-01 |
| JP2542787B2 (en) | 1996-10-09 |
| DE4226949A1 (en) | 1994-02-17 |
| ZA935914B (en) | 1995-02-13 |
| EP0583717B1 (en) | 1998-03-18 |
| KR940004326A (en) | 1994-03-14 |
| AU656479B2 (en) | 1995-02-02 |
| DE59308274D1 (en) | 1998-04-23 |
| AU4453493A (en) | 1994-05-05 |
| ATE164229T1 (en) | 1998-04-15 |
| JPH06213898A (en) | 1994-08-05 |
| EP0583717A1 (en) | 1994-02-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Schade et al. | Bound iron and unsaturated iron-binding capacity of serum; rapid and reliable quantitative determination | |
| US4292296A (en) | Diagnostic method | |
| GB2275774A (en) | Stabilized composition of troponin | |
| EP0154276B1 (en) | Specific binding assay employing anti-g6pdh as label | |
| EP0928421B1 (en) | Standard solution for the determination of thyroid function | |
| Sölling | Polymeric forms of free light chains in serum from normal individuals and from patients with renal diseases | |
| US4018883A (en) | Thyroxine (T4) radioimmunoassay | |
| US5342788A (en) | Method and standard solution for the determination of thyroxine (T4) or triiodothyronine (T3) | |
| US4588695A (en) | Determination of unsaturated iron-binding capacity | |
| US4052504A (en) | Assay for thyroxine binding globulin | |
| US4081525A (en) | Radioimmunoassay of plasma steroids | |
| Cheung et al. | Use of polyethylene glycol in separating bound from unbound ligand in radioimmunoassay of thyroxine. | |
| JPH0667960B2 (en) | Method for separating analyte bound to binding protein and sample preparation reagent for measuring vitamin B12 in serum | |
| CA1334925C (en) | Process for the determination of thyroxine and a suitable standard solution therefor | |
| Bojesen et al. | Water-phase palmitate concentrations in equilibrium with albumin-bound palmitate in a biological system. | |
| US4350659A (en) | Stabilization of sensitive biologically active intermediate metabolites such as folic acid derivatives | |
| AU656479B2 (en) | Method and standard solution for the determination of thyroxine (T4) or triiodothyronine (T3) | |
| US5576219A (en) | Standard solutions for determination of thyroxine-binding capacity in serum | |
| US6387711B1 (en) | Liquid intact parathyroid hormone (PTH) standards | |
| WERNER et al. | Rapid radioimmunoassay for both T4 and T3 in the same sample of human serum | |
| US4066410A (en) | Thyroid hormone assay | |
| USRE32098E (en) | Radioimmunoassay for measurement of thyroxine (T4) and triiodothyronine (T3) in blood serum | |
| US6610291B2 (en) | Ready-to-use ristocetin cofactor test reagent possessing long-term stability | |
| US4399228A (en) | Polate competitive protein binding assay | |
| US4431741A (en) | Hypothyroid control serum |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |